The American Journal of Pathology, Vol. 169, No. 3, September 2006
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2006.060180
Immunopathology and Infectious Diseases
B and T Lymphocytes Are the Primary Sources of
RANKL in the Bone Resorptive Lesion of Periodontal
Disease
Toshihisa Kawai,* Takashi Matsuyama,†
Yoshitaka Hosokawa,* Seicho Makihira,*
Makoto Seki,*‡ Nadeem Y. Karimbux,§
Reginaldo B. Goncalves,* Paloma Valverde,¶
Serge Dibart,储 Yi-Ping Li,** Leticia A. Miranda,*
Cory W.O. Ernst,* Yuichi Izumi,† and
Martin A. Taubman*
From the Departments of Immunology * and Cytokine Biology,**
The Forsyth Institute, Boston, Massachusetts; the Department of
Oral Medicine, Infection, and Immunity,§ Harvard School of
Dental Medicine, Boston, Massachusetts; the Department of
General Dentistry,¶ Tufts University School of Dental Medicine,
Boston, Massachusetts; the Department of Periodontology,储
Goldman School of Graduate Dentistry, Boston University,
Boston, Massachusetts; the Department of Periodontology,†
Kagoshima University, Kagoshima, Japan; and R&D Division,‡
Mitsubishi Pharma, Tokyo, Japan
Receptor activator of nuclear factor-B (RANKL)-mediated osteoclastogenesis plays a pivotal role in inflammatory bone resorption. The aim of this study was to identify the cellular source of RANKL in the bone resorptive
lesions of periodontal disease. The concentrations of
soluble RANKL, but not its decoy receptor osteoprotegerin, measured in diseased tissue homogenates were
significantly higher in diseased gingival tissues than in
healthy tissues. Double-color confocal microscopic
analyses demonstrated less than 20% of both B cells and
T cells expressing RANKL in healthy gingival tissues. By
contrast, in the abundant mononuclear cells composed
of 45% T cells, 50% B cells, and 5% monocytes in diseased gingival tissues, more than 50 and 90% of T cells
and B cells, respectively, expressed RANKL. RANKL production by nonlymphoid cells was not distinctly identified. Lymphocytes isolated from gingival tissues of patients induced differentiation of mature osteoclast cells
in a RANKL-dependent manner in vitro. However, similarly isolated peripheral blood B and T cells did not
induce osteoclast differentiation, unless they were activated in vitro to express RANKL; emphasizing the osteoclastogenic potential of activated RANKL-expressing
lymphocytes in periodontal disease tissue. These results
suggest that activated T and B cells can be the cellular
source of RANKL for bone resorption in periodontal
diseased gingival tissue. (Am J Pathol 2006, 169:987–998;
DOI: 10.2353/ajpath.2006.060180)
Periodontal disease (or periodontitis) is an inflammatory
lesion that is accompanied by soft tissue destruction and
bone resorption in the tooth-supporting structures. A positive correlation between the occurrence of disease and
elevated serum antibody response to the oral bacteria
colonizing the gingival crevice1–3 suggests the involvement of an immune response to the multiple bacteria in
the onset and development of periodontal disease. In
general, immune responses to bacteria are considered to
be a host protective mechanism to pathogenic bacteria.
However, despite the elevated IgG antibody response to
certain disease-associated bacteria colonizing the periodontal crevice, inflammation and/or bone resorption
proceed in the periodontitis lesions. The question is
posed as to whether immune response to periodontal
bacteria is protective, or otherwise pathogenic, in the
context of periodontal disease.
The fundamental cytokine system that underlies bone
resorption processes is dependent on the osteoclast differentiation, activation, and survival factor, receptor activator of nuclear factor-B (RANKL), and its soluble decoy
receptor osteoprotegerin (OPG).4 – 6 Involvement of immune cells in the course of bone resorption has been
demonstrated by the expression of RANKL on activated T
cells.7 RANKL expressed by T cells, as well as by osteoblasts and bone marrow stromal cells, triggers signaling
in osteoclast precursor cells that elicits the differentiation
into their mature form.8 OPG is expressed ubiquitously by
many types of cells and tissues, and it counterregulates
Supported by the National Institute of Dental and Craniofacial Research
(grants DE-03420, DE-14551, and DE-15722).
Accepted for publication June 6, 2006.
Address reprint requests to Martin A. Taubman, Department of Immunology, The Forsyth Institute, 140 The Fenway, Boston, MA 02115. E-mail:
mtaubman@forsyth.org.
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the excessive bone loss by antagonizing the RANKLbinding to its receptor RANK.9 The paradigm of osteoclast differentiation regulation is based on the RANKL/
OPG ratio expressed in the microenvironment
surrounding osteoclast precursor cells.10
An active periodontal lesion is characterized by the
prominent infiltration of B cells11,12 and T cells.13,14 Although the T cells infiltrating the inflamed gingival tissues
express activation markers such as CD45RO15 or
CD29,16 functional roles of these activated T cells are not
completely clear. We demonstrated that adoptive transfer
of RANKL⫹, antigen-specific T cells can induce bone
loss in rat periodontal tissue that received local injection
of the T-cell antigen.17,18 Teng and colleagues19 reported that adoptive transfer of an Actinobacillus actimomycetemcomitans-specific human T cell line isolated from
patients with aggressive (juvenile) periodontal disease
could induce significant periodontal bone loss in NOD/
SCID mice orally inoculated with A. actinomycetemcomitans every 3 days.19 Although the latter report showed
that systemic administration of OPG-Fc could reduce
periodontal bone loss, it is not clear whether the transferred human T cells or bystander cells that might be
secondarily stimulated by the transferred human T cells
are the source of RANKL. It has been reported that
RANKL produced by B cells is responsible for the devastating bone resorption in multiple myeloma.20 Activation of B cells in vitro can induce expression of RANKL,
but these cells are deficient in production of OPG.21
Recently, we reported that antigen-specific activated B
cells can induce periodontal bone resorption in a rat
model.22 However, it is unclear if B cells accumulating in
periodontal diseased tissue express RANKL.
To determine the cellular source of RANKL in bone
resorptive periodontitis, enzyme-linked immunosorbent
assay (ELISA) and double-color confocal microscopic
analyses were used. Results of ELISA demonstrated that
soluble RANKL (sRANKL) production was significantly
elevated in gingival tissues with periodontal disease
compared to healthy gingival tissues. Confocal microscopic analyses showed that both T cells and B cells, but
not monocytes or fibroblasts, are the cellular source of
RANKL in the bone resorptive lesion of periodontal disease. Importantly, RANKL expressed by periodontal T
cells and B cells appeared to be the osteoclastogenic
functional component, as determined by in vitro RANKLdependent osteoclast differentiation assays.
Materials and Methods
Patients
Patients diagnosed with chronic periodontitis [n ⫽ 32
(including three smokers), 12 males and 21 females;
average age, 46.9 years; range of ages, 33 to 67 years]
were otherwise systemically healthy patients. These patients had periodontal bone resorption diagnosed by Xray examination, bleeding on probing, and clinical gingival crevice probe depths of greater than 3 mm at the
diseased site. Informed consents from all patients were
obtained before sample collection. The diseased gingival
tissue lesions and healthy tissues were sampled during
surgical treatment. Healthy gingival tissues were collected from patients with gingival crevice depth of equal
to or less than 3 mm and with no X-ray indication of bone
loss, at surgery for tooth restorative purposes including
crown lengthening [n ⫽ 12 (including one smoker), five
males and seven females; average age, 43.4 years;
range of ages, 25 to 72 years].
Reverse Transcriptase-Polymerase Chain
Reaction (RT-PCR)
Total RNA was extracted from gingival tissues and RTPCR was performed as previously described.23,24 Primer
pairs for human RANKL and OPG were as follows:
RANKL forward primer, 5⬘-TCAGAAGATGGCACTCACTG-3⬘ and RANKL reverse primer, 5⬘-AACATCTCCCACTGGCTGTA-3⬘ (PCR product size 879 bp),25 OPG
forward primer, 5⬘-GCCCTGACCACTACTACACA-3⬘ and
OPG reverse primer, 5⬘-TCTGCTCCCACTTTCTTTCC-3⬘
(PCR product size 736 bp). Total RNA isolated from
gingivae (1 g) was synthesized to cDNA. The resulting
cDNA was subject to PCR by amplifying 30 cycles for
RANKL or for OPG along with -actin23 as an internal
control (94°C for 30 second, 60°C for 1 minute, 72°C for
1 minute, and final elongation at 72°C for 10 minutes).
PCR products were separated in 1.7% agarose gels and
stained with ethidium bromide.
Preparation of Gingival Tissue Homogenates
Gingival tissues were homogenized with a Dounce glass
homogenizer in phosphate-buffered saline supplemented with 0.05% Tween 20, phenylmethyl sulfonyl fluoride (1 mmol/L; Sigma, St. Louis, MO), and protease
inhibitor cocktail (Sigma), as published with slight modification.26 For ELISA, the concentration of sRANKL, interleukin (IL)-1, and OPG in the tissue homogenates or
culture supernatants were measured with ELISA kits for
human soluble RANKL (sRANKL) (Peprotech, Rocky Hill,
NJ) and DuoSet ELISA for human IL-1, OPG, IL-10,
IL-12 p70, and GM-CSF (R&D Systems, Minneapolis,
MN). The method for detection of IgG response to oral
bacteria has been previously published.27,28 To detect
anti-bacterial IgG levels in the gingival homogenates, the
following formalin-fixed bacteria were used as antigen in
ELISA: A. actinomycetemcomitans strain Y4 (American
Type Culture Collection no. 43718), Fusobacterium nucleatum str. 25586, Eikenella corrodens str. 23834, Porphyromonas gingivalis str. W83, and Prevotella intermedia str.
25611.
Culture of Peripheral Blood Mononuclear
Cells (PBMCs)
PBMCs were collected from healthy patients under informed consent agreement (n ⫽ 4; 28- to 40-year-old
males; average age, 34.5 years). The mononuclear cell
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(MC) fraction was separated from blood by gradient centrifugation using Histopaque-1077 (Sigma) and incubated in RPMI 1640 supplemented with 10% fetal bovine
serum, 2-mercaptoethanol, L-glutamine, and penicillin
and streptomycin. T cells were enriched from PBMCs by
glass-wool and nylon-wool column purification.24 B cells
were enriched from PBMCs by negative selection using
magnetic beads coated with a mixture of anti-CD3 monoclonal antibody (mAb) (OKT3: American Type Culture
Collection, Rockville, MD; UCTH1: R&D Systems), CD4
(OKT4), CD8 (OKT8), and CD28 (CD28.2; BD Pharmingen, San Diego, CA). The enriched T cells or B cells were
stimulated with immobilized antibodies on a 96-well culture plate with mouse mAb to CD3 (OKT3) and CD28
(CD28.2) or mAb to CD40 (5C3, BD Pharmingen) and
goat polyclonal anti-human IgM (Serotec, Oxford, UK),
respectively.
Immunofluorescent Laser-Scanning
Confocal Microscopy
Basic fluorescent staining technique was performed as
previously reported.24 Sectioned tissues mounted on
glass slides were fixed with 2% paraformaldehyde. T
cells, B cells, and monocytes were stained with the
mouse monoclonal antibodies to CD3 (UCTH1), CD20
(H1; BD Pharmingen), and CD14 (3C10; American Type
Culture Collection), respectively, followed by fluorescein
isothiocyanate-conjugated anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) as a secondary reagent. RANKL expression was monitored by
biotinylated-OPG-Fc followed by Texas Red-Avidin (Invitrogen, Carlsbad, CA). To block FcRn and Fc␥RII,
which can bind to monomeric ␥ chain of human IgG-Fc,
human IgG was prereacted with some of the gingival
tissue sections. OPG-Fc was generously provided by Dr.
Colin Dunston (Amgen, Thousand Oaks, CA) under mutual material transfer agreement and was conjugated with
biotin using EZ-Link sulfo-NHS-biotin (Pierce, Rockford,
IL). A human IgG-Fc fusion protein (L6) conjugated with
biotin was used as a negative control. After washing, the
cover glass was set on the sample with Fluoromount-G
mounting medium (Southern Biotechnology, Birmingham,
AL). The staining pattern was analyzed by 0.3 m sequential optical sectioning at ⫻400 or ⫻1000 magnification with a Leica TCS/SP-2 laser-scanning confocal microscope (Leica, Wetzlar, Germany).
Osteoclast Differentiation Assay
MCs were isolated from patient gingival tissue biopsies
as previously described.23 Patient gingival MCs, fresh
PBMCs, stimulated PB T cells, or stimulated PB B cells in
vitro were fixed with formalin. Human PB CD14⫹ monocytes were isolated from PBMCs using a magnetic beadbased monocyte-negative isolation kit (Dynal Biotech,
Oslo, Norway). In some experiments, patient gingival
MCs were incubated in 96-well plates for 3 hours, and
nonadherent cells were separated from the adherent
cells on the culture plate. The adherent gingival MCs
were ⬃80% CD14⫹ monocytes, whereas nonadherent
gingival MCs contained less than 2% CD14⫹ cells by
immunostaining. The resulting adherent cells in the wells
and nonadherent gingival MCs were fixed with formalin
and were used in the following co-culture system. The
fixed human lymphocytes were co-cultured in 96-well
plates with the mouse osteoclast precursor cell line
MOCP-529 or PB CD14⫹ monocytes in the presence of
M-CSF (10 ng/ml; Peprotech). Recombinant human
RANKL (50 ng/ml; R&D Systems) was added to the
MOCP-5 culture, as a positive control. To assess the
involvement of RANKL in the osteoclast differentiation,
OPG-Fc (10 g/ml) was added to antagonize RANKL in
some cultures. The culture medium was changed every 3
days by replacing half of the volume. After 6 to 8 days of
culture for MOCP-5, or 14 to 16 days of culture for PB
CD14⫹ monocytes, when multinuclear osteoclast-like
cells are observed by phase-contrast microscopy, cells
were fixed with 5% formalin-saline. Differentiated osteoclasts were identified as tartrate-resistant acid phosphatase (TRAP)-positive cells with three or more nuclei as
described previously.30 The TRAP⫹ cells with more than
three nuclei were counted as osteoclasts using phase
contrast microscopy and expressed as cell number/well
of 96-well plates. The bone resorption activity of osteoclasts was evaluated by a pit formation assay using a
calcium phosphate-coated tissue culture vessel system
(Biocoat Osteologic System; BD Biosciences, San Jose,
CA) or dentin disks (Alpco Diagnostics, Windham, NH)
according to the manufacturers’ instructions.
Results
Detection of sRANKL and OPG Proteins in
Gingival Tissue Homogenates
The concentration of sRANKL protein in gingival tissue
was significantly elevated (P ⬍ 0.02, t-test) in diseased
tissues as compared to healthy gingival tissues (Figure
1A). In addition, there was a positive correlation between
the level of sRANKL in tissues and the depth of the
gingival crevice where the tissues were sampled (Figure
1B). Differences in OPG concentration between diseased
and healthy tissues were not statistically significant (Figure 1C). The proinflammatory cytokine IL-1 was monitored in the same groups of gingival tissues (Figure 1D),
as an indicator of the degree of inflammation in each
tissue. All gingival tissues from diseased lesions expressed significantly higher IL-1 amounts than the
healthy gingival tissues (Student’s t-test, P ⬍ 0.05). We
also evaluated the concentrations of the osteoclastogenesis inhibitory cytokines, IL-10, IL-12 p70, and GM-CSF
and the osteoclast precursor chemoattractant factor,
MIP-1␣, compared to healthy tissues. However, although
the diseased gingival tissues had higher mean concentrations of each of the four factors, none of the differences
were statistically significant. Compared to healthy patients’ antibody levels, the tissue homogenates of patient
gingival tissues also demonstrated significantly higher
IgG antibody levels to three of five periodontal disease-
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Figure 2. RANKL and OPG mRNA expression in gingival tissues. RT-PCR was
performed to detect mRNA expression of RANKL and OPG in the total RNA
extracted from whole gingival tissue samples. Expression of RANKL mRNA
was detected in 67% of diseased periodontal tissues (solid bars, four of six),
whereas no expression of RANKL mRNA was observed in the healthy gingival tissues tested (open bars, zero of four), whereas OPG mRNA message
was observed from healthy patients (one of four) and from periodontal
disease tissues (two of six). The relative intensity of each mRNA message in
the acrylamide gel was scanned by densitometer using the AlphaImage
analysis system and expressed as relative signal intensity (RSI) in the histograms (B, RANKL; C, OPG).
Figure 1. Protein concentrations of RANKL, OPG, and IL-1 in gingival
tissues. Protein concentrations of RANKL and OPG in gingival tissues were
measured by ELISA. Gingival tissue samples were homogenized in the
presence of proteinase inhibitors (healthy, n ⫽ 3; disease, n ⫽ 11). The
concentration of each protein, RANKL (A and B), OPG (C), and IL-1 (D), is
expressed as pg per mg of gingival tissue (pg/mg). *Significantly higher than
healthy control by Student’s t-test (P ⬍ 0.05). B: The concentrations of
RANKL were positively correlated with the gingival pocket depth where the
biopsy was sampled. **Positive correlation (n ⫽ 14, P ⬍ 0.01).
associated bacteria examined in this study (F. nucleatum,
P. gingivalis, and P. intermedia; data not shown).
Expression of RANKL and OPG mRNA in
Gingival Tissue
RT-PCR was performed to detect mRNA expression of
RANKL and OPG in the gingival tissue RNA samples
(Figure 2). RANKL mRNA expression was detected from
67% of the diseased gingival tissues (four of six) examined, whereas no expression of RANKL mRNA was observed from the healthy gingival tissues tested (zero of
four). OPG mRNA message was observed in tissue from
periodontally diseased patients (two of six) and one
healthy subject (one of four).
RANKL Expression by T and B Lymphocytes in
Gingival Tissue
To identify the cell types that express RANKL in inflamed
gingival tissue, double-color confocal microscopy (Figure 3) was used after staining for RANKL (red) and lymphocyte-specific CD makers (green). Images of RANKL
and CD marker-positive cells were merged in the com-
puter system, and double-positive cells were displayed
as yellow staining. Gingival tissues from periodontitis lesions demonstrated marked expression of RANKL (Figure 3, A–C). The cellular infiltrates in the diseased gingival tissue were predominantly T (Figure 3A) and B
lymphocytes (Figure 3B), with few CD14⫹ monocytes
(Figure 3C). RANKL was expressed in these cellular infiltrates, especially by T cells and by B cells, and to a
much lesser extent by monocytes. Although most B cells
were positive for RANKL staining, not all T cells expressed RANKL. Very few lymphocytes expressing
RANKL were present in healthy gingival tissues (Figure
3D). RANKL expression by other nonlymphoid cell types,
such as fibroblasts, was not distinctly observed.
The numbers of CD3⫹ and CD20⫹ lymphocytes infiltrating the gingival tissues were significantly higher than
those in the healthy gingival tissues, whereas the number
of CD14⫹ cells did not show a remarkable increase (Figure 4). Importantly, the percentage of CD14⫹ cells to the
number of total lymphocytes remained low irrespective of
the gingival condition of disease/healthy (19% in healthy
versus 5% in disease gingival tissue; Figure 4). These
data strongly indicate that T cells and B cells are a much
more significant source of RANKL than CD14⫹ cells in
the diseased gingival tissues.
The percentage of RANKL⫹ cells in the population of T
cells or B cells was also determined (Figure 5) based on
counts of the images obtained in the confocal microscopic analyses (Figure 3). The percentage of RANKLexpressing T cells (Figure 5A) or B cells (Figure 5B)
demonstrated a positive correlation with the depth of the
gingival crevice. Importantly, in the diseased gingival
tissues, RANKL⫹ cells were greatly elevated compared
to the healthy tissues, and a significantly higher percent-
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Figure 3. Confocal microscopic analyses of expression of RANKL in gingival tissues. Using double-color confocal microscopy, RANKL-expressing cells were
identified in the gingival tissue of a periodontal disease patient (A–C) and in healthy gingival tissue (D). CD3 (A and D), CD20 (B), or CD14 (C) expression was
indicated by staining with various specific mouse mAbs followed by fluorescein isothiocyanate anti-mouse IgG (left column). RANKL expression was detected
by OPG-biotin followed by streptavidin-Texas Red (middle column). Both specific CD marker-stained cells and RANKL⫹-stained cells were doubly exposed and
expressed as yellow (merged; right column). CD20⫹ and CD14⫹ cells in healthy tissue (not shown) were as few as CD3⫹ cells in healthy gingival tissue (D). Scale
bars ⫽ 20 m.
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Figure 4. Numbers of lymphocytes in the gingival tissues. The total number
of CD3⫹, CD20⫹, and CD14⫹ cells in a confocal microscopic field of each
tissue section was counted (⫻400, average cell number of at least three
sections was enumerated as the number of cells per sample) and compared
between healthy gingival tissues (open column) and disease gingival tissues
(filled column). Mean cell number ⫾ SD is shown. *Significantly higher than
healthy gingival tissue by Student’s t-test (P ⬍ 0.01).
age of B cells expressed RANKL (94 ⫾ 5% SD) than T
cells (51 ⫾ 18% SD) (Figure 5C).
Activation of Peripheral Blood T Cells and B
Cells Induces sRANKL Production
To investigate whether naı̈ve or activated lymphocytes
express RANKL, peripheral blood T cells or B cells from
healthy patients were stimulated with immobilized antiCD3 and anti-CD28 mAbs or with immobilized anti-CD40
and/or anti-IgM, respectively (Figure 6). The stimulation
via CD3/CD28 or CD40/BCR (B-cell receptor; anti-IgM)
induced proliferation and sRANKL expression by the purified T cells or B cells [Figure 6, A and C (T cells) and B
and D (B cells)]. Although production of OPG by the
activated T cells or B cells was also monitored (by
ELISA), the level of OPG production was lower than the
detection limit (4 pg/ml). An oral bacterial stimulus (fixed
A. actinomycetemcomitans) also activated peripheral
blood lymphocytes to produce both sRANKL and cellular
RANKL from T cells and B cells (examined by confocal
microscopy, not shown). The activated T cells and B cells
isolated from peripheral blood were able to induce osteoclast (TRAP⫹, multinucleated) cell differentiation in a
RANKL-dependent manner (Figure 6E), whereas activated CD14⫹ monocytes did not induce such osteoclastogenesis (Figure 6E). Of note, most published literature
also supports the finding that CD14⫹ cells express of
RANK but not RANKL.31,32 These data suggested that T
cells and B cells that express RANKL in gingival tissues
are probably in the activated form and that these activated T and B cells are the major source of RANKL.
Evaluation of the Osteoclast Differentiation
Function of RANKL Expressed by Gingival
Lymphocytes
To evaluate the functional aspects of RANKL expressed
by MCs isolated from the diseased gingival tissue, an in
vitro differentiation assay was outperformed using the
osteoclast precursor cell line MOCP-5 (Figure 7A) or
Figure 5. Expression of percentage of RANKL⫹ T cells or B cells in the
gingival tissues. After computer capture of the confocal microscopic image of
each gingival section (⫻400), the percentage of yellow cells (double positive
for CD3/RANKL or CD20/RANKL) of the total number of green cells (CD3⫹
or CD20⫹ ) were calculated and are shown on scatter plots (A, CD3⫹ T cells;
B, CD20⫹ B cells), according to the depth of gingival crevice from which the
samples were collected (open triangles, healthy patients; filled triangles,
periodontal disease patients). Both RANKL⫹ T cells and RANKL⫹ B cells
showed positive correlation with the depth of gingival crevice (T cells: n ⫽
34, r ⫽ 0.507, P ⬍ 0.01; B cells: n ⫽ 15, r ⫽ 0.702, P ⬍ 0.01). C: Percentage
of RANKL⫹ T cells or B cells of the total T cells or B cells in the gingival
tissues classified by the depth of the gingival crevice (GC depth) is shown.
The data shown in A and B were converted to histograms, presented as
mean ⫾ SD of the percentage of RANKL⫹ T cells or B cells. *Statistically
significant by Student’s t-test (P ⬍ 0.0001). Note: the percentage of RANKL⫹
CD14⫹ monocytes/total CD14⫹ monocytes (not shown) was 15.3 ⫾ 16.8 and
59.5 ⫾ 39.9 in healthy and disease gingival tissues, respectively, showing no
statistical differences by t-test.
peripheral blood CD14⫹ monocytes (Figure 7B). Both
MOCP-5 cells and CD14⫹ monocytes differentiated into
TRAP⫹ multinucleated cells in response to recombinant
human sRANKL in the presence of M-CSF. The isolated
diseased gingival MCs as well as recombinant RANKL
induced TRAP⫹ multinucleated cells (Figure 7A). However, only nonadherent diseased gingival MCs, but not
adherent gingival MC cells, induced RANKL-dependent
osteoclastogenesis (Figure 7B), indicating that any
RANKL expression that might have been detected on
CD14⫹ cells (Figure 3C) is not functionally active. The
morphological appearance of the differentiated MOCP-5
cell line is shown [Figure 8, A (sRANKL) and C (patient
gingival MC)]. Induction of TRAP⫹ multinucleated cells
was abolished by the addition of OPG-Fc into the culture
[Figure 7 and Figure 8, B (sRANKL ⫹ OPG-Fc) and D
(patient gingival MC ⫹ OPG-Fc)], demonstrating that
RANKL expressed by MCs from diseased tissues can
induce RANKL-dependent osteoclast differentiation. The
pit formation assay was performed to assess the functional capabilities of differentiated multinucleated cells
(Figure 8, E–J). Resorption pits shown as positively
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Figure 6. Activation of peripheral blood T cells or B cells induces sRANKL
expression. Purified T cells isolated from a healthy subject PBMCs (2 ⫻
105/well) were activated for 7 days with immobilized anti-CD3 (⫻CD3) and
anti-CD28 (⫻CD28) mAbs, and proliferation (A) and sRANKL production (C)
were measured by [3H]thymidine incorporation assay and by ELISA, respectively. Purified B cells isolated from the PBMCs (2 ⫻ 105/well) were also
incubated in a 96-well plate for 7 days with immobilized anti-CD40 (⫻CD40),
anti-IgM (⫻IgM), and anti-CD40 and anti-IgM antibodies, and proliferation
(B) and sRANKL production (D) were measured by the same method as for
T cells. E: Osteoclastogenesis induced by the activated peripheral blood T
cells, B cells, and CD14⫹ monocytes is shown. The cultured T cells or B cells
under the conditions shown in A or B were harvested and fixed on day 7.
After activation of these CD14⫹ monocytes with fixed A. actinomycetemcomitans for 4 days, CD14⫹ monocytes produced IL-12 (143 ⫹ 23 pg/ml),
but not RANKL or OPG. The fixed T cells, B cells, or CD14⫹ monocytes were
co-cultured with MOCP-5 (104/well) in the presence of M-CSF (10 ng/ml) as
described under Materials and Methods. The number of TRAP-positive
multinuclear cells induced in each well was counted and expressed as
mean ⫾ SD of triplicate determinants. *Significantly higher than medium
alone by Student’s t-test (P ⬍ 0.05). Other PBMC samples isolated from one
periodontal diseased and two orally healthy patients showed similar results.
stained by toluidine blue (indicated by arrows) were observed in the disks from wells in which MOCP-5 cells
were co-cultured with sRANKL or MCs from diseased
gingival tissue (Figure 8, F and G, respectively). The
formation of resorption pits induced by MOCP-5 co-cultured with MCs from the diseased tissue was inhibited by
the presence of OPG (not shown). The pit formation assay using a calcium-phosphate-coated tissue culture
Figure 7. Osteoclast differentiation assay using the MOCP-5 osteoclast precursor cell line or human peripheral blood CD14⫹ monocytes. A: MOCP-5
osteoclast precursor cells were co-cultured with human recombinant RANKL,
fresh nonstimulated PBMCs, or gingival MCs isolated from periodontal disease patients. PBMCs and gingival MCs were prefixed with formalin before
co-culture with MOCP-5 (104/well). B: CD14⫹ monocytes isolated from
PBMCs of a healthy subject (2 ⫻ 104/well) were co-cultured with human
recombinant RANKL or with fixed adherent or nonadherent gingival MCs
isolated from periodontal disease patient. All culture medium contained
recombinant human M-CSF (10 ng/ml) in the presence or absence of OPG-Fc
(10 g/ml) (both A and B). Half of the total culture medium was exchanged
every 3 days. On day 8 (A) or on day 16 (B), the cultures were fixed with
formalin, and TRAP staining was outperformed. The numbers of cells with
more than three nuclei demonstrating TRAP⫹ staining were counted in each
well under phase contrast microscopy. Similar results of TRAP⫹ cell induction were obtained using the MCs isolated from two different patients’
gingival tissues. *Significantly elevated compared to the control MOCP-5 (A);
significantly elevated compared to human CD14⫹ monocytes cultured in
medium alone without OPG-Fc (B) by Student’s t-test (P ⬍ 0.01).
vessel system (Figure 8, H–J) showed similar results to
the assay using the dentin disks (Figure 8, E–G). The
MOCP-5 co-cultures with MCs from diseased tissue
showed resorption pits (Figure 8J, white clear area, indicated by arrows) as well as MOCP-5 incubated with
sRANKL (Figure 8I, arrows), compared to the negative
control of MOCP-5 cultured in medium alone, which did
not show such resorption pits (Figure 8H).
Discussion
The present study of the bone resorptive lesions of periodontal disease tissues demonstrated that, not only T
cells, but also B cells, are major sources of RANKL. The
concentrations of sRANKL and IL-1 examined in the
gingival tissue homogenates were significantly elevated
in the diseased gingival tissues compared to healthy
tissues, whereas OPG protein production was not significantly higher in diseased tissues than healthy tissues.
Overexpression of OPG in transgenic mice and RANK
gene knockout mice develop severe osteopetrosis33,34
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Immune Cells, Source of RANKL in Periodontitis
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AJP September 2006, Vol. 169, No. 3
and OPG gene knockout mice demonstrate osteopenia.35 The ratio of RANKL to OPG is normally increased in
a bone resorptive lesion characterized by extensive osteoclastic activity.36 Therefore nearly exclusive production of RANKL and the lack of production of OPG by
lymphocytes seem to contribute to the increased RANKL
to OPG ratio in the bone resorptive periodontitis lesions.
Key studies showed that RANKL gene knockout mice not
only develop osteopetrosis but are also deficient in both
T and B lymphocytes,10,37 implying a RANKL-associated
linkage between T and B cells and bone resorption.
Another study using a SCID-Hu mouse model indicated
that human T cells can express RANKL in response to
certain oral bacteria.19 RANKL protein expression has
been demonstrated by T cells isolated from periodontitis
gingival tissue by flow cytometry.38 Consistent with our
findings that diseased gingival tissue contains much
more sRANKL than healthy tissue (Figure 1A) and that the
concentration of sRANKL is greater in deeper gingival
crevices (Figure 1B) is the recent report that the ratio of
sRANKL to OPG in gingival crevice fluid (GCF) is elevated in periodontitis patients compared to healthy patients.39 Therefore, the findings presented herein that
RANKL is expressed by T cells and also B cells and that
a higher percentage of B cells express RANKL than T
cells in periodontitis tissue provides information quite
relevant to the elucidation of bone resorptive factors in
periodontal disease.
We have recently demonstrated that activated antigenspecific B cells can induce periodontal bone resorption in
a RANKL-dependent manner using a rat periodontitis
model.22 B cells primed in vivo with specific antigen (A.
actinomycetemcomitans) up-regulated RANKL expression in response to in vitro stimulation with antigen. The
adoptive transfer of these donor antigen-primed B cells to
congenitally athymic recipient rats that received simultaneous gingival injection with antigen resulted in periodontal bone resorption in conjunction with elevated IgG antibody response to A. actinomycetemcomitans.22 This
periodontal bone resorption was abrogated by OPG-Fc
local injection.22 Accumulation of plasma cells and B
cells in the disease lesion11,12 and elevated IgG antibodies to periodontal bacteria in the serum1,2,28 and gingival
crevice fluid40 are distinct features of periodontitis. However, the nature of B cells in the context of periodontal
bone resorption has not been clearly elucidated until our
previous study.22 Herein, we revealed that B cells, in
addition to T cells, are primary cellular sources of bone
destructive factor by immunohistological studies of human periodontal disease lesions.
In our previous studies of the rat periodontal disease
model using adoptive transfer of T-clone cells, antigenspecific stimulation of the T-clone cells enhanced expression of RANKL mRNA to a greater degree than expression of OPG mRNA as determined by RT-PCR, and
resulted in local periodontal bone resorption.17 Sakata
and colleagues41 reported that dental mesenchymal
cells produce OPG and enhance OPG production in
response to proinflammatory factors such as IL-1 or
tumor necrosis factor (TNF)-␣. Nagasawa and colleagues38 showed that lipopolysaccharide stimulation of
gingival fibroblasts induced OPG expression and inhibited differentiation of monocytes to osteoclast cells. Although the ELISA system in the present study detected
OPG protein production in both healthy and diseased
gingival tissues, mRNA message for OPG was not detected in healthy gingival tissue as measured by RT-PCR.
Because the same RT-PCR system demonstrated the
mRNA message of -actin and RANKL, nonspecific
mRNA degradation by RNases during the sampling process was excluded as the cause of such discrepancy
between OPG mRNA and OPG protein expression. Furthermore, the difference between in vivo (present study)
and in vitro cultures of dental mesenchymal cells41 may
cause the discrepancy in OPG mRNA detection. In the
present study, stimulation of peripheral blood T cells and
B cells in vitro did not produce detectable levels of OPG
irrespective of stimulation (data not shown). Therefore,
the OPG production in both healthy and diseased gingival tissues may be attributed, at least, to fibroblasts in
periodontal tissue that are capable of producing physiologically relevant amounts of OPG, maintaining the homeostasis of healthy periodontal bone remodeling.
Although few studies investigated the mechanism underlying osteoclast recruitment to the bone resorption
lesion, MIP-1␣ and other CCR1 -chemokines seem to be
associated with the osteoclast migration as well as osteoclast formation and bone resorption.42,43 With respect to
the chemotaxis of osteoclast precursors, the homing
mechanism of such cells to gingival tissues remains unclear. In the present study, MIP-1␣ concentration in diseased gingival tissues was not significantly different from
healthy tissues.
In metastatic bone disease, the ␣-chemokine IL-8 has
been implicated as a stimulator of osteoclastogenesis
and bone resorption.44 However, IL-8 is an important
chemoattractant factor to recruit neutrophils to gingival
tissues because an innate immune reaction and defect in
neutrophil function is thought to be a pathogenic mechanism in periodontal disease.45 It is important to clarify
Figure 8. TRAP⫹ multinuclear cells induced from MOCP-5 osteoclast precursors by recombinant RANKL and patient’s gingival MCs. MOCP-5 osteoclast precursor
cells were co-cultured with human recombinant RANKL or with gingival MCs isolated from the patient’s periodontal disease lesion. Patient’s gingival MCs fixed
with formalin (104/well) were applied to the MOCP-5 culture. MOCP-5 cells were cultured on the oval-shape coverslip in a 96-well plate. Medium that contained
M-CSF (10 ng/ml) was exchanged (50% of total culture volume) every 3 days. On day 8, the cultures were stopped, and the cells were fixed with formalin. The
MOCP-5 cells adherent on the coverslip were reacted with TRAP reagent and mounted on glass slides. The figures demonstrate the appearance of TRAP staining
of MOCP-5 co-cultured for 8 days with recombinant human RANKL (50 ng/ml) (A), recombinant human RANKL plus OPG-Fc (10 g/ml) (B), fixed patient’s
gingival MCs isolated from a diseased lesion (C), and fixed patient’s gingival MCs isolated from a diseased lesion plus OPG-Fc (10 g/ml) (D). The bone-resorptive
activities of TRAP⫹ multinuclear cells were examined using dentin disks (E–G) and the Osteologic system (H–J). The MOCP-5 was cultured in a medium
supplemented with M-CSF (10 ng/ml) for 8 days in the following different conditions; medium alone (E, H), in the presence of recombinant RANKL (50 ng/ml)
(F, I), in the presence of patient’s fixed gingival MCs (G, J). The arrows in A and C indicate the TRAP⫹ multinuclear cells. The arrows in E–J indicate the
resorption pits formed by TRAP⫹ multinuclear cells. Of note, toluidine blue stains resportion pits dark brown (F, G), while the resorption pits in the Osteologic
system are demonstrated as white clear areas (I, J).
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Kawai et al
AJP September 2006, Vol. 169, No. 3
the role of IL-8 in periodontal bone resorption because T
cells and B cells, which are found to be the major sources
of RANKL, are attracted by -chemokines including
MIP-1␣ but not by IL-8.
In addition to RANKL and OPG, other cytokines including M-CSF, IL-1, IL-6, IL-11, IL-17, and TNF-␣ have been
reported to support osteoclast formation by up-regulating
RANKL expression, promoting osteoclast differentiation
in a RANKL-dependent or perhaps in a RANKL-independent manner.5,46 – 49 By contrast to these osteoclastogenesis-promoting cytokines, IL-10 is reported to inhibit osteoclast differentiation.50 Although the role of GM-CSF is
controversial, this cytokine seems to decrease the osteoclastogenesis induced by bone marrow stromal cells.51
IL-12 and IL-18 are indicated as osteoclast inhibitory
cytokines through mechanisms dependent on GM-CSF52
or IFN-␥.53 In the present study, an inflammatory cytokine, IL-1, measured in gingival tissues was significantly
elevated in the diseased tissues as was RANKL (Figure 1,
A and E), whereas IL-12, IL-10, and GM-CSF levels were
not significantly different in the diseased gingival tissues
compared to the healthy tissues. Because IL-1 is downstream of TNF-␣-induced osteoclastogenesis,54,55 it is
conceivable that elevated IL-1 may reflect the TNF-␣
produced in periodontal tissues. The involvement of
TNF-␣ in the periodontal bone loss was demonstrated in
a primate model of periodontal disease; ie, soluble receptors to IL-1 or to TNF-␣, respectively, can significantly
inhibit the inflammatory response and bone loss.56 TNF-␣
can cause osteoclastogenesis in a RANKL-independent
manner,46 and also synergistically increase RANKL-dependent osteoclast differentiation.54 However, OPG-Fc
mediated nearly complete inhibition of in vitro osteoclastogenesis from MOCP-5 and from human peripheral
blood CD14⫹ monocytes (Figure 7, A and B). These
findings seemed to exclude the possible involvement of
membrane-bound TNF-␣ and other secondary osteoclast-stimulatory components expressed on activated T
cells and/or B cells.
It is well documented that generic inflammation in the
synovial cavity can lead to the secondary expression of
RANKL and other osteoclast differentiation factors.55 We,
however, hypothesize that the mechanism underlying
periodontal bone resorption is different from the mechanism for rheumatoid arthritis, because the healthy oral
cavity is constantly exposed to bacterial inflammatory
mediators, such as lipopolysaccharide or peptidoglycan,57 whereas the normal synovial cavity is free from
bacterial components. Nonetheless, healthy gingival tissue is free from bone resorption, whereas mitogenic stimulation associated with bacteria may cause inflammation
in synovial tissues. Our hypothesis is that generic inflammation, per se, is not the cause of bone resorption in the
periodontal tissues. This hypothesis is supported by gingivitis, another form of periodontal inflammation,58 that
does not exhibit bone resorption. Our rat periodontal
disease model also illustrated that antigen-specific T-cell
and B-cell activation in the inflamed gingival tissues is
necessary for the induction of periodontal bone loss, and
simple inflammation induced by lipopolysaccharide
alone does not cause periodontal bone resorption.22,30
Therefore RANKL expression by activated lymphocytes
may be a prerequisite to develop osteoclast differentiation in the gingival tissues having pre-existing
inflammation.
It is conceivable that not only lymphocytes but also
osteoblasts may be involved in increases of the RANKL to
OPG ratio in the inflamed gingival tissue of periodontitis.
The present study did not evaluate osteoblasts, which are
also a potential RANKL source in periodontal disease
tissue. IL-1 and TNF-␣ appear to be present in the inflamed gingival tissues59 and are able to induce RANKL
expression by osteoblast cells. Despite the potential involvement of other factors in the bone destruction process, prominent expression of RANKL by B and T cells in
the periodontal disease lesion seems to play a primary
role in the augmentation of bone resorption processes in
this disease.
Acknowledgments
We thank Dr. Colin Dunstan (Amgen Inc., Thousand
Oaks, CA) for OPG-Fc; Dr. Zedonis Skobe, Dr. Rick A.
Rogers, Ms. Jean Lai, and Ms. Elke Pravda for confocal
microscopy support; and Dr. Takashi Yanaoka and Mr.
Tadahiko Kounoike for some support of this study.
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